The present invention relates to the treatment of ischemic wounds, for example, where the injury results from lack of oxygen due to poor circulation such as in the diseases diabetes, scleroderma, and the like, by the administration of relaxin. The present invention also relates to the promotion of angiogenesis.
Mature human relaxin is a hormonal peptide of approximately 6000 daltons known to be responsible for remodelling the reproductive tract before parturition, thus facilitating the birth process. This protein appears to modulate the restructuring of connective tissues in target organs to obtain the required changes in organ structure during pregnancy and parturition. See, Hisaw, F. L., Proc. Soc. Exp. Biol. Med., 23: 661-663 (1926); Schwabe, C., et al., Biochem. Biophys. Res. Comm., 75: 503-570 (1977); James, R. et al., Nature, 267: 544-546 (1977). A concise review of relaxin was provided by Sherwood, D. in The Physiology of Reproduction, Chapter 16, xe2x80x9cRelaxinxe2x80x9d, Knobil, E. and Neill, J., et al. (eds.), (Raven Press Ltd., New York), pp. 585-673 (1988). Circulating levels of relaxin are elevated for the entire nine months of pregnancy and drop quickly following delivery.
While predominantly a hormone of pregnancy, relaxin has also been detected in the non-pregnant female as well as in the male. Bryant-Greenwood, G. D., Endocrine Reviews, 3: 62-90 (1982) and Weiss, G., Ann. Rev. Physiol., 46:43-52 (1984).
Relaxin has been purified from a variety of species including porcine, murine, equine, shark, tiger, rat, dogfish and human, and shows at least primary and secondary structural homology to insulin and the insulin-like growth factor. In the human, relaxin is found in most abundance in the corpora lutea (CL) of pregnancy. However, specific nuclei in the brain have relaxin receptors and other nuclei contain messenger RNA for relaxin. Several nuclei with cells bearing relaxin receptors are found in the area of the hypothalamus.
Two human gene forms have been identified, (H1) and (H2). Hudson, P., et al., Nature, 301: 628-631 (1983); Hudson, P., et al., The EMBO Journal, 3: 2333-2339 (1984); and U.S. Pat. Nos. 4,758,516 and 4,871,670. Only one of the gene forms (H2) has been found to be transcribed in CL. It remains unclear whether the (H1) form is expressed at another tissue site, or whether it represents a pseudo-gene. When synthetic human relaxin (H2) and certain human relaxin analogs were tested for biological activity, the tests revealed a relaxin core necessary for biological activity as well as certain amino acid substitutions for methionine that did not affect biological activity. Johnston, et al., in Peptides: Structure and Function, Proc. Ninth American Peptide Symposium, Deber, C. M., et al. (eds.) (Pierce Chem. Co. 1985).
Methods of making relaxin are also described in U.S. Pat. No. 4,835,251 and in co-pending U.S. Ser. Nos. 07/908,766 (PCT US90/02085) and 08/080,354 (PCT US94/0699). Methods of using relaxin in cardiovascular therapy and in the treatment of neurodegenerative diseases are described in U.S. Pat. No. 5,166,191 and in U.S. Ser. No. 07/902,637 (PCT US92/06927). Certain formulations of human relaxin are described in allowed U.S. Ser. No. 08/050,745.
Recombinant human relaxin (H2) in currently in Phase I human clinical trials in scleroderma patients. Scleroderma is a disease involving an imbalance in tissue reformation giving rise to the overproduction of collagen, and ultimately resulting in swelling and hardening of the skin (and affected organs).
Vascular Endothelial Growth Factor (VEGF) has also been localized in situ in the corpus luteum (CL) of pregnancy, as well as the placenta and the endometrium. See Sharkey et al., J. Reprod. Fert. 99:609-615 (1993); Li et al. Growth Factors 22:277-282 (1994); Phillips et al. Endocrinology 127:965-967 (1990). VEGF, highly conserved glycoprotein secreted by macrophages, exhibits a potent ability to induce new vessel growth in vivo. VEGF is mitogen specific for endothelial cells and can induce both endothelial cell migration and serine and metalloproteinase expression (for review, see Thomas, K. A., J. Biol. Chem. 271:603-606 (1996). The strongest sites of VEGF expression are the fetal and maternal macrophages. Besides its proposed role in promoting new vessel growth during pregnancy, VEGF has also been proposed to be involved in persistent and dysregulated vessel growth in pathological conditions such as tumor metastasis, diabetic retinopathy, and rheumatoid arthritis.
In one aspect, the invention relates to a method of promoting angiogenesis in a mammal in need thereof by administering a therapeutically effective amount of relaxin. In a preferred embodiment, relaxin is administered in an amount sufficient to maintain a serum concentration of at least about 1 ng/ml. In a further preferred embodiment the relaxin is recombinant human relaxin (H2).
In another aspect, the invention relates to the treatment of infections or ischemic wounds by administering a therapeutically effective amount of relaxin. In a particularly preferred embodiment, the infection or ischemic wound is one where injury has resulted from lack of oxygen due to poor circulation.
In yet another aspect of the invention, there is provided a method of using relaxin for the manufacture of a medicant for the treatment of an infection or ischemic wound, or for the manufacture of a medicant for the promotion of angiogenesis. In preferred versions of these embodiments, the relaxin is recombinant human relaxin (H2).
Definitions and General Parameters
As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.
xe2x80x9cOptionalxe2x80x9d or xe2x80x9coptionallyxe2x80x9d means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.
The term xe2x80x9ctreatmentxe2x80x9d or xe2x80x9ctreatingxe2x80x9d means any therapeutic intervention in a mammal, including:
(i) prevention, that is, causing the clinical symptoms not to develop;
(ii) inhibition, that is, arresting the development of clinical symptoms; and/or
(iii) relief, that is, causing the regression of clinical symptoms.
The term xe2x80x9ceffective amountxe2x80x9d means a dosage sufficient to provide treatment for the disease state being treated. This will vary depending on the patient, the disease and the treatment being effected.
The term xe2x80x9crelaxinxe2x80x9d means human relaxin, including intact full length relaxin or a portion of the relaxin molecule that retains biological activity [as described in U.S. Pat. No. 5,023,321, preferably recombinant human relaxin (H2)] and other active agents with relaxin-like activity, such as Relaxin Like Factor (as described in U.S. Pat. No. 5,911,997 at SEQ ID NOS: 3 and 4, and column 5, line 27-column 6, line 4), relaxin and portions that retain biological activity analogs and portions that retain biological activity (as described in U.S. Pat. No. 5,811,395 at SEQ ID NOS: 1 and 2, and column 3, lines 16-40), and agents that competitively displace bound relaxin from a receptor. Relaxin can be made by any method known to those skilled in the art, preferably as described in U.S. Pat. No. 4,835,251 and in co-pending U.S. Ser. Nos. 07/908,766 (PCT US90/02085) and 08/080,354 (PCT US94/0699).
The invention is based, in part, on the surprising discovery that relaxin promotes angiogenesis in an in vivo assay, as described more fully below by way of working examples. Specifically, relaxin was found angiogenic in both a rabbit corneal injection protocol and by the matrigel subcutaneous insert vascularization protocol.
Also reported herein is the novel discovery that relaxin induces secretion of a potent angiogenic factor, Vascular Endothelial Growth Factor (xe2x80x9cVEGFxe2x80x9d), in the monocyte-like cell line THP-1. This finding further broadens the scope of relaxin""s known biological activity. Since macrophages are known to play a key role in angiogenesis, both during and outside of pregnancy, relaxin""s potential regulatory role straddles both.
In the THP-1 cell line, relaxin stimulates the expression of at least 3 of the 4 isoforms of VEGF: the 121, the 165, and the 189 amino acid isoforms. Although all forms are reportedly bioactive, the 121 and 165 amino acid forms are secreted while the larger molecules remain associated with the extracellular matrix unless enzymatically released. This stimulation of relaxin expression occurred at the transcriptional level, even in the presence of cycloheximide, indicating that no de novo protein synthesis was required. Additionally, results presented herein indicate that the induction of VEGF by relaxin in THP-1 cells may be mediated by cAMP and protein kinase C. This finding is consistent with the known role of cAMP in the stimulation of VEGF expression in other cell types. See, for example, Claffey et al., J. Biol. Chem. 267:16317-16322 (1992), and Garrido et al., Growth Factors 8:109-117 (1993). The rapid increase in VEGF transcripts following relaxin treatment of THP-1 cells is similar to that seen in preadipocytes following forskolin stimulation (Garrido et al., supra). Indeed, the rapidity of the induction and the lack of cycloheximide effect may suggest a pathway common to THP-1 cells and preadipocytes.
Utility
Relaxin is useful for promoting angiogenesis and treating infections and ischemic wounds (e.g., poorly healing ischemic ulcers) characteristic of diseases, such as diabetes and scleroderma, involving poorly vascularized disease sites and macrophage associated inflammation. Macrophages are one of the most important sources of angiogenic factors. It has surprisingly been discovered that certain macrophage lines contain relaxin binding sites. It has also surprisingly been discovered that relaxin promotes angiogenesis in vivo.
Testing
In vitro activity for relaxin binding to macrophages is determined using p32 labeled relaxin binding sites.
In vivo activity for angiogenesis is determined by the rabbit corneal injection protocol and by the matrigel subcutaneous insert vascularization protocol.
Administration
Relaxin is administered at a therapeutically effective dosage, e.g., a dosage sufficient to promote angiogenesis and/or provide treatment for the above-referenced disease states.
Administration of relaxin can be via any of the accepted modes of administration for agents that serve similar utilities, preferably by systemic administration.
While human dosage levels for promoting angiogenesis have yet to be optimized for relaxin, generally, a daily dose is from about 0.1 to 500.0 xcexcg/kg of body weight per day, preferably about 6.0 to 200.0 xcexcg/kg, and most preferably about 12.0 to 100.0 xcexcg/kg. Generally it is sought to obtain a serum concentration of relaxin approximating or greater than normal circulating levels in pregnancy, i.e., 1.0 ng/ml, such as 0.5 to 50 ng/ml, preferably 1.0 to 20 ng/ml. In the ongoing clinical trials, dosages of about 6.0 xcexcg/kg, 12.0 g/kg and 50 xcexcg/kg have respectively resulted in serum concentrations of about 1.8 ng/mlxc2x10.3, 3.6 ng/mlxc2x10.6, and 11.8 ng/mlxc2x11.6. Thus, for administration to a 70 kg person, the dosage range would be about 7.0 xcexcg to 3.5 mg per day, preferably about 42.0 xcexcg to 2.1 mg per day, and most preferably about 84.0 to 700.0 xcexcg per day. The amount of relaxin administered will, of course, be dependent on the subject and the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician.
In employing relaxin for treatment of the above conditions, any pharmaceutically acceptable mode of administration can be used. Relaxin can be administered either alone or in combination with other pharmaceutically acceptable excipients, including solid, semi-solid, liquid or aerosol dosage forms, such as, for example, tablets, capsules, powders, liquids, gels, suspensions, suppositories, aerosols or the like. Relaxin can also be administered in sustained or controlled release dosage forms (e.g., employing a slow release bioerodable delivery system), including depot injections, osmotic pumps (such as the Alzet implant made by Alza), pills, transdermal and transcutaneous (including electrotransport) patches, and the like, for prolonged administration at a predetermined rate, preferably in unit dosage forms suitable for single administration of precise dosages. The compositions will typically include a conventional pharmaceutical carrier or excipient and relaxin. In addition, these compositions may include other active agents, carriers, adjuvants, etc. Generally, depending on the intended mode of administration, the pharmaceutically acceptable composition will contain about 0.1% to 90%, preferably about 0.5% to 50%, by weight of relaxin, the remainder being suitable pharmaceutical excipients, carriers, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. The formulations of human relaxin described in U.S. Ser. No. 08/050,745 are particularly preferred.
Parenteral administration is generally characterized by injection, either subcutaneously, intradermally, intramuscularly or intravenously, preferably subcutaneously. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, solubility enhancers, and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, cyclodextrins, and the like.
The percentage of relaxin contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the needs of the subject. However, percentages of active ingredient of 0.01% to 10% in solution are employable, and will be higher if the composition is a solid which will be subsequently diluted to the above percentages. Preferably the composition will comprise 0.2-2% of the relaxin in solution.
A more recently devised approach for parenteral administration employs the implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained. Various matrices (e.g., polymers, hydrophilic gels, and the like) for controlling the sustained release, and for progressively diminishing the rate of release of active agents such as relaxin are known in the art. See, U.S. Pat. No. 3,845,770 (describing elementary osmotic pumps); U.S. Pat. Nos. 3,995,651, 4,034,756 and 4,111,202 (describing miniature osmotic pumps); U.S. Pat. Nos. 4,320,759 and 4,449,983 (describing multichamber osmotic systems referred to as push-pull and push-melt osmotic pumps); and U.S. Pat. No. 5,023,088 (describing osmotic pumps patterned for the sequentially timed dispensing of various dosage units).
Formulations of relaxin may also be administered to the respiratory tract as a nasal or pulmonary inhalation aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients. In such a case, the particles of the formulation may advantageously have diameters of less than 50 microns, preferably less than 10 microns. See, e.g., U.S. Pat. No. 5,364,838, which discloses a method of administration for insulin that can be adapted for the administration of relaxin in the present invention.